Dear Monika,

Here are my first thoughts:

1. Do you use several siRNAs specific to your target? Maybe your single siRNA is just ineffective (in case)?

2. Can you try other transfection reagent? At least RNAiMAX (that's what we are working with) is stated to work on HepG2.

Maybe this handbook will be helpful:

http://www.lifetechnologies.com/content/dam/LifeTech/global/life-sciences/RNAi/PDFs/rnai%20sourcebook%20final.pdf.

You may also like to browse four guidelines found at: http://www.lifetechnologies.com/ru/en/home/references/ambion-tech-support/rnai-sirna/general-articles/-sirna-design-guidelines.html

Ain't no ad. We are following it all by ourselves. If someone suggests something better or in addition, I'll personally be grateful.

Good luck,

Peter

1. First try different transfection reagents

2. Do twice transfection

Dear Monika,

I have used RNAimax and lipofectamine2000 in HepG2 and they work well with knockdown after 1 day of treatment. Perhaps you need to troubleshoot the following:

1. is the siRNA vial shipped right and new?

2. the methodology used

3. may be you need to repeat your siRNA transfection one more time. how many times have you tried?

I have used a concentration of 10nM in HepG2 and it works well.

Good luck!

Thanks all.

Hi Peter, I am using eSiRNA from sigma. But I used their transfection reagent , so that might be the reason.I will see if I can get RNAi MAX and will try.

Hi Chunxing Yang , I have already tried thrice, but can not see any noticable knockdown. I will try different transfection reagent.

Hi Abbas Raza, I need to SiRNA vial. Can I check by native PAGE? or which protocol do you follow for checking quality of SiRNA.

I have never experienced this problem. Checking on denaturing agarose gel may help but you need to load 200 ng of RNA atleast and siRNA is way too expensive for such studies.

Can I ask you the siRNA details, which gene are you targeting? Does it knock down all the variants?

I have had success with Lipofectamine2000 in HepG2 cells. Do you have any other siRNAs that you know work well? I usually transfect a known, effective siRNA into new cells to establish the best transfection conditions. Once you've optimized transfection, you can transfect your siRNA of interest. If it still doesn't work, it's an ineffective siRNA.

I am using SiRNA against receptor SCARB1.

I am not using any positive control at the moment. I will probably order one to optimize.

Thanks

hello dear Monica,

I suggest that you follow the protocol for hepG2 siRNA transfection introduced by life technologies company. In this protocol reverse transfection of hepG2 cell is recommended.  

Hi Monica,

Sorry for branching out from your question. But since I am using the similar esirna1 product for my gene of interest from Sigma, i had this curiosity to understand how did you know the molar concentration of your sirna i.. 30nM or so...because the product doesnt mention it, it gives the conc in ng/ul. How did you convert it to nM? Since we don't know the sequences either. Can you help?